Bengal tigers (Panthera tigris tigris) and Indian leopards (Panthera pardus fusca) are widespread across the Indian subcontinent and form a major part of apex predators in the forest ecosystem. However, both species are endangered, and their fragile populations could be threatened by the introduction of pathogenic agents. In the present study, archived biological samples of big cats collected from different protected areas and zoological parks in central India were subjected to molecular and histological analysis for canine distemper virus (CDV) infection. Preserved biological samples were processed for molecular detection of CDV using nucleoprotein gene primers. Nucleotide sequencing and BLAST analysis of the positive samples demonstrated a close similarity to the CDV isolates from several wild carnivore hosts. Immunohistochemistry performed on formalin-fixed tissues showed that CDV antigens were diffusely distributed in the tissues. Histopathological observations were consistent across all CDV-positive tigers and leopards. Histopathology revealed interstitial pneumonia, interstitial nephritis, lymphoid depletion in the spleen, hepatic inflammation, degeneration of transitional epithelium in the bladder, and white matter demyelination, gliosis, and neuronal necrosis in the brain. Our findings revealed that CDV is prevalent in the big cats in central India. Therefore, it is imperative to develop multifaceted protocols to screen for such emerging infectious diseases in field samples.